FIGURE 4.

Regulation of A-CAT and MHCK A by Thr825. A, the Coomassie Blue-stained SDS gel shows wild-type MHCK A (WT) and the MHCK A T825A and T825E mutants purified from D. discoideum as described under “Experimental Procedures.” B, the kinase activities of MHCK A (WT) and the MHCK A T825A and T825E mutants were assayed as described under “Experimental Procedures.” Mutation of Thr⁸²⁵ severely inhibited the kinase activity of MHCK A. Activities are reported as a percentage of MHCK A activity. Error bars represent the standard deviation. C, the top line shows the sequence of the A-CAT C-tail and indicates the residues deleted to generate the Δ3 and Δ6 constructs. The second line shows the sequence of the MHCK B C-tail (residues 326–354) that was fused to Leu⁸⁰⁵ of A-CAT to generate the A-CAT-BT chimera. The conserved Gly-Thr sequence is underlined. The kinase (left panel) and ATPase (right panel) activities of wild-type A-CAT (WT), the Δ3 and Δ6 constructs, and the A-CAT-BT chimera (BT) were determined from time courses performed as described under “Experimental Procedures.” The C-tail of MHCK B rescues the activity of the truncated A-CAT. Activities are reported as a percentage of wild-type A-CAT activity. Error bars represent the standard deviation.